Article

The Rockefeller University Press J. Exp. Med. 2017 Vol. 214 No. 8 2387–2404https://doi.org/10.1084/jem.20162152

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IntroductIonVan Furth and colleagues suggested an adult monocytic or-igin of all macrophages, but it is now clear that many tis-sue-resident macrophages, including alveolar macrophages in the lung, are self-renewing populations that arise from fetal progenitors and require minimal input from circuiting adult monocytes in a healthy environment (Guilliams et al., 2013; Yona et al., 2013; Scott et al., 2014; Kopf et al., 2015; van

de Laar et al., 2016). In response to macrophage depletion, monocytes are recruited to the lung, where the microen-vironment shapes them into cells that closely resemble tis-sue-resident alveolar macrophages (Landsman and Jung, 2007; Hashimoto et al., 2013; Lavin et al., 2014; Gibbings et al., 2015). Accordingly, during homeostasis, the lung harbors at least one and perhaps two ontologically distinct populations of alveolar macrophages, referred to as tissue-resident alveo-lar macrophages (TR-AMs) and monocyte-derived alveolar macrophages (Mo-AMs). Our current understanding of the role of alveolar macrophages is almost exclusively based on studies conducted in healthy animals during homeostasis, but

Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibro-sis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. during the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tis-sue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span

Alexander V. Misharin,1* Luisa Morales-Nebreda,1* Paul A. Reyfman,1* Carla M. Cuda,2 James M. Walter,1 Alexandra C. McQuattie-Pimentel,1 Ching-I Chen,1 Kishore R. Anekalla,1 Nikita Joshi,1 Kinola J.N. Williams,1 Hiam Abdala-Valencia,2 Tyrone J. Yacoub,3 Monica Chi,1 Stephen Chiu,1,4 Francisco J. Gonzalez-Gonzalez,1 Khalilah Gates,1 Anna P. Lam,1 Trevor T. Nicholson,1 Philip J. Homan,2 Saul Soberanes,1 Salina Dominguez,2 Vince K. Morgan,2 Rana Saber,2 Alexander Shaffer,2 Monique Hinchcliff,2 Stacy A. Marshall,5 Ankit Bharat,1,4 Sergejs Berdnikovs,6 Sangeeta M. Bhorade,1 Elizabeth T. Bartom,4 Richard I. Morimoto,7 William E. Balch,8 Jacob I. Sznajder,1 Navdeep S. Chandel,1 Gökhan M. Mutlu,9 Manu Jain,1 Cara J. Gottardi,1 Benjamin D. Singer,1 Karen M. Ridge,1 Neda Bagheri,2 Ali Shilatifard,4 G.R. Scott Budinger,1** and Harris Perlman3**

1Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine and 2Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL

3Department of Chemical and Biological Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL4Division of Thoracic Surgery, Department of Surgery, Feinberg School of Medicine, 5Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, and 6Division of Allergy and Immunology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL

7Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL8Department of Molecular Medicine, The Scripps Research Institutes, La Jolla, CA9Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL

© 2017 Misharin et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org /terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /).

*A.V. Misharin, L. Morales-Nebreda, and P.A. Reyfman contributed equally to this paper.

**G.R.S. Budinger and H. Perlman contributed equally to this paper.

Correspondence to G.R.S. Budinger: s-buding@northwestern.edu; Harris Perlman: h-perlman@northwestern.edu

Abbreviations used: IM, interstitial macrophage; Mo-AM, monocyte-derived alveolar macrophage; PCA, principal-component analysis; RIPK, receptor-interacting protein kinase; RNA-seq, RNA sequencing; TR-AM, tissue-resident alveolar macrophage.

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Monocyte-derived alveolar macrophages drive lung fibrosis | Misharin et al.2388

the differentiation of monocytes into alveolar macrophages may differ in the local microenvironment resulting from tissue injury or fibrosis.

It has long been recognized that depletion of circulat-ing monocytes (e.g., using Ccr2−/− mice or via the systemic administration of liposomal clodronate) ameliorates fibrosis severity in the lung and other organs (Moore et al., 2001; Gibbons et al., 2011; Wynn and Vannella, 2016). To explain these findings, investigators have suggested that during injury, monocytes rapidly differentiate into Mo-AMs and that both TR-AMs and Mo-AMs are polarized toward a profibrotic or “M2” phenotype in the fibrotic lung (Zhou et al., 2014). This “macrophage polarization” model is based on limited evidence and is conceptually incomplete (Xue et al., 2014; Nahrendorf and Swirski, 2016). For example, even in the healthy lung, alveolar macrophage differentiation is a slow process that unfolds over a time course longer than most fibrosis models (weeks), raising the possibility that incom-pletely differentiated alveolar macrophages rather than “po-larized” alveolar macrophages contribute to fibrosis (Lavin et al., 2014; Gibbings et al., 2015). Furthermore, the relative contributions of Mo-AMs compared with TR-AMs to the development of fibrosis is not known. Finally, it is not clear whether alveolar macrophages are reconstituted after injury through the differentiation of recruited Mo-AMs or by the proliferation of TR-AMs and, if the former, whether these ontologically distinct populations are functionally different. Answers to these questions are important for the design of monocyte/macrophage–targeted therapies. In particular, monocyte-depletion strategies are limited by the requirement for a continuous supply of monocyte-derived cells for ho-meostasis in the gut and other tissues; therefore, it is critical to know whether selectively targeting Mo-AMs after they have lost canonical monocyte markers can ameliorate fibrosis. Similarly, strategies that nonselectively deplete both TR-AMs and Mo-AMs might paradoxically worsen fibrosis by induc-ing further recruitment of monocytes and threaten tissue ho-meostasis through the loss of TR-AM function (Janssen et al., 2011; Gibbings et al., 2015).

To address these questions, we developed a novel lineage tracing system in mice to unambiguously identify Mo-AMs and TR-AMs during the development of fibrosis and over the subsequent life span of the animal. We used this system to show that lung fibrosis was ameliorated when Mo-AMs were driven to necroptosis during their differentiation with-out affecting monocytes, thereby establishing a causal link between Mo-AMs and the pathobiology of fibrosis. In con-trast, depletion of TR-AMs before the induction of lung fibrosis did not alter fibrosis severity. In 14-mo-old untreated mice, >95% of AMs were TR-AMs, but in mice treated with bleomycin at 4 mo of age, the alveolar macrophage pool was comprised of both TR-AMs and Mo-AMs, suggesting that a subpopulation of Mo-AMs persists after the resolution of fibrosis. Using transcriptomic profiling of monocyte-derived populations collected over the course of fibrosis and its reso-

lution, we show that monocyte to macrophage differentiation unfolds slowly over weeks. Genes identified by others as caus-ally linked to the development of fibrosis were most highly expressed in Mo-AMs early during their differentiation and were progressively down-regulated as Mo-AMs differentiated into mature alveolar macrophages. TR-AMs and Mo-AMs showed significant differences in profibrotic gene expres-sion during fibrosis; however, 10 mo after the injury, these differences were no longer evident. Human homologues of many of the profibrotic genes expressed in Mo-AMs during bleomycin-induced fibrosis were differentially expressed in flow-sorted alveolar macrophages obtained from the lungs of patients with fibrotic lung disease. These results reveal remarkable heterogeneity in alveolar macrophage function during lung fibrosis with important implications for the de-sign of targeted therapy.

resuLtsIn the absence of tissue injury, monocyte to macrophage dif-ferentiation is thought to be regulated by epigenetic changes in response to factors present in the lung microenvironment (Lavin et al., 2014). Depletion of circulating monocytes using Ccr2−/− mice or the administration of liposomal clodronate reduces fibrosis severity, implicating monocyte-derived cells in the development of fibrosis (Moore et al., 2001; Gautier et al., 2012; Lavin et al., 2014; Gibbings et al., 2015). How-ever, we know little about the importance of monocytes or monocyte-derived cells after they leave the circulation. We have previously shown that 5 d after administration of bleo-mycin, the number of interstitial macrophages (identified as CD64+Silgec F−) was increased, whereas the number of alve-olar macrophages (CD64+Siglec F+) was reduced. In contrast, during the fibrotic phase (day 21 post–bleomycin administra-tion), the number of interstitial macrophages decreased and the number of alveolar macrophages increased because of the appearance of alveolar macrophages characterized by lower expression of Siglec F (Siglec Flow; Fig. 1, A and B; Misharin et al., 2013). To determine whether this new population of cells represented an expansion of TR-AMs or the recruitment of Mo-AMs, we developed a lineage tracing system that allows us to distinguish alveolar macrophage ontogeny during injury and over the subsequent life span of the animal (Fig. 1 C). Using this system, we showed that the increase in alveolar macrophages during fibrosis was completely attributable to monocyte-derived cells (i.e., Mo-AMs). Furthermore we found that differential expression of Siglec F reliably distin-guished Mo-AMs and TR-AMs over the course of bleomy-cin-induced fibrosis (Fig. 1, B, E, and F; Misharin et al., 2013), and we used this gating strategy (Fig. S1) to identify TR-AMs and Mo-AMs in subsequent studies.

We next sought to determine whether Mo-AMs, TR-AMs, or both contributed to the development of fibrosis. First, to target Mo-AMs, we used mice with targeted deple-tion of capsase-8 in macrophages (Cuda et al., 2014, 2015). Caspase-8 is a cysteine protease that serves the dual function

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of activating the extrinsic apoptotic pathway in response to death ligands and suppressing receptor-interacting protein kinase (RIPK)–mediated necroptosis during development and inflammation (Lu et al., 2014; Salvesen and Walsh, 2014). We deleted Casp8 from different monocyte/macrophage populations using two independent Cre drivers (CD11cCre

Casp8flox/flox and LysMCreCasp8flox/flox) that both efficiently target alveolar macrophages. We then administered intratra-cheal bleomycin to Casp8flox/flox, CD11cCreCasp8flox/flox, and LysMCreCasp8flox/flox mice and compared the severity of fibro-sis after 21 d. We found that although a substantial fraction of control Casp8flox/flox mice died in response to bleomycin, sur-vival was improved in both CD11cCreCasp8flox/flox and LysMCre

Casp8flox/flox animals (Fig. 2 A). Furthermore, the severity of fibrosis was significantly worse in Casp8flox/flox animals than in CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox animals (Fig. 2, B–D). We confirmed these results in another model of lung fibrosis induced by the intratracheal administration

of an adenoviral vector encoding an active form of TGF-β (Lam et al., 2014; Morales-Nebreda et al., 2015). Mice with macrophage-targeted deletion of Casp8 showed attenuated Ad-TGF-β–induced fibrosis (Fig. S2, A–C). One model for alveolar macrophage development involves the sequential differentiation of monocytes into interstitial macrophages (IMs), which in turn differentiate into alveolar macrophages, although it is also possible that monocytes differentiate di-rectly into alveolar macrophages. We observed reduced num-bers of Mo-AMs and increased numbers of IMs in CD11cCre

Casp8flox/flox and LysMCreCasp8flox/flox animals when compared with Casp8flox/flox animals (Fig. 2, E and F; and Fig. S2, D and E), suggesting that Mo-AMs are lost during the differentia-tion of IMs or monocytes.

Our findings could be explained by the induction of necroptosis in Mo-AMs as they differentiate in the lungs of Casp8-deficient mice during fibrosis. If so, we reasoned that deletion of RIPK3 in CD11cCreCasp8flox/flox and LysMCre

Figure 1. tr-AMs are depleted during lung fibrosis and replaced with Mo-AMs. (A and B) The alveolar macrophage pool is expanded 21 d after the intratracheal administration of bleomycin when fibrosis is maximal because of the appearance of a novel population of cells characterized by lower expres-sion of Siglec F (n = 5 per group, paired t test; data are shown as mean ± SEM; ***, P < 0.001). (C) Schematic explaining the technique for the generation of bone marrow chimeras with thoracic shielding and busulfan and representative dot plots reflecting chimerism in peripheral blood monocytes and alveolar macrophages in the lung 8 wk after irradiation and bone marrow transfer. Whole-body irradiation leads to complete replacement of recipient monocytes and alveolar macrophages with donor-derived cells (top). Irradiation with thoracic shielding protects alveolar macrophages but results in incomplete chi-merism among circulating monocytes (middle). The addition of busulfan conditioning postirradiation allows complete elimination of recipient’s monocytes while preserving alveolar macrophages (bottom). (D) Percentage of monocytes and alveolar macrophages of donor and recipient origin 8 wk after the generation of chimeras (n = 3–5 per group; data are shown as mean ± SEM and are representative of >100 mice). (E) The new population of alveolar mac-rophages that emerges during bleomycin-induced fibrosis (CD45+CD11b+/−CD11c+CD64+SiglecFlow) is monocyte derived. Representative contour plots show expansion of the alveolar macrophage pool; numbers indicate percentage of the parent population (gated on singlets/live/CD45+ cells). (F) Expansion of the alveolar macrophage pool during bleomycin-induced lung fibrosis is caused by recruitment of Mo-AMs, whereas the number of TR-AMs is decreased (n = 5 per group, paired t test; data are shown as mean ± SEM and are representative of more than five independent experiments; **, P < 0.01; ****, P < 0.0001).

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Casp8flox/flox animals should restore the Mo-AM population and “rescue” fibrosis. To test this hypothesis, we generated Casp8flox/flox, CD11cCreCasp8flox/floxRIPK3−/−, and LysMCre

Casp8flox/floxRIPK3−/− mice and treated them with bleomy-cin. The loss of RIPK3 restored the population of Mo-AMs to control levels, and the severity of fibrosis in Casp8flox/flox, CD11cCreCasp8flox/floxRIPK3−/−, and LysMCreCasp8flox/flox

RIPK3−/− mice was similar to that seen in Casp8flox/flox an-imals (Fig.  2, A–F). These data suggest that RIPK3 is acti-

vated during monocyte to macrophage differentiation. To exclude a functional role for RIPK3 in alveolar macro-phage differentiation, as has been suggested in bone mar-row–derived cells (Dannappel et al., 2014; Moriwaki et al., 2014; Vlantis et al., 2016), we flow-sorted Mo-AMs from Casp8flox/flox, CD11cCreCasp8flox/floxRIPK3−/−, and LysMCre

Casp8flox/floxRIPK3−/− mice and measured their transcrip-tomes using RNA sequencing (RNA-seq). We found that only a small number of genes were differentially expressed

Figure 2. necroptosis of Mo-AMs attenuates bleomycin-induced lung fibrosis. (A–D) Mice with deficiency of Casp8 in alveolar macrophages have (A) improved survival (16–101 mice per group; combined data from 10 independent experiments; Mantel-Cox log-rank test), (B) lower levels of collagen, (C) improved static lung compliance, and (D) less extent histological fibrosis compared with controls, whereas simultaneous deletion of RIPK3 rescues bleomycin-induced lung fibrosis in CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice (n = 9–45 mice per group, data expressed as mean ± SEM, one-way ANO VA with Dunnett’s test for multiple comparisons; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). (E and F) Protection from lung fibrosis is associated with the loss of Mo-AMs, but not TR-AMs, in Casp8-deficient mice, whereas RIPK3 deficiency rescues Mo-AMs (n = 4–5 mice per group; data are expressed as mean ± SEM; one-way ANO VA with Dunnett’s test for multiple comparisons; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; the experiment was performed at least two times). (G and H) The loss of RIPK3 does not alter the response of alveolar or interstitial macrophages to fibrosis in CD11cCreCasp8flox/floxRIPK3−/− and LysMCreCasp8flox/floxRIPK3−/− mice. Volcano plot showing number of differentially expressed genes (red, FDR q value < 0.05; n = 2–5 mice per group). DEG, differentially expressed genes.

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in all investigated cell types, suggesting RIPK activation is largely dispensable for alveolar macrophage differentiation and fibrosis in this model (Fig. 2, G and H).

Both CreLysM and CreCD11c should effectively target both Mo-AMs and TR-AMs; therefore, we wondered whether the protection we observed in the CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice be partially attributed to de-letion of Casp8 in TR-AMs. Both CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice had normal numbers of TR-AMs in comparison to Casp8flox/flox mice (Fig. 3 A). Surprisingly, the levels of Casp8 mRNA in TR-AMs from naive CD11cCre

Casp8flox/flox mice were similar to those in the Casp8flox/flox mice (Fig.  3  B). This suggested to us that RIPK-mediated necroptosis might occur as fetal monocytes differentiate into TR-AMs during the early postnatal period, thereby providing a selection pressure favoring TR-AMs that escape recombi-nation. Consistent with this hypothesis, Casp8 was efficiently deleted in TR-AMs from the RIPK3−/− animals (Fig. 3 B). To confirm that the loss of Casp8 prevented the differentiation of

monocytes into Mo-AMs, we compared the ability of bone marrow from Casp8-deficient donors to reconstitute the al-veolar macrophage pool in the presence or absence of RIPK3. Compared with wild-type bone marrow, bone marrow from the Casp8-deficient donors on a wild-type background inef-ficiently repopulated the alveolar macrophage pool, as shown by ongoing recruitment of alveolar macrophages with low levels of Siglec F expression in CD11cCreCasp8flox/flox relative to Casp8flox/flox mice (Fig. 3, C and D). Deletion of RIPK3 (CD11cCreCasp8flox/floxRIPK3−/−) restored the monocyte to alveolar macrophage differentiation, as indicated by normal levels of Siglec F. We confirmed these findings by demon-strating a reduced ability of bone marrow from CD11cCre

Casp8flox/flox relative to Casp8flox/flox mice to reconstitute the alveolar macrophage, but not circulating monocyte, pool in 1:1 completive chimera experiments (Fig. 3 E).

To exclude the possibility that the partial loss of Casp8 we observed in TR-AMs from CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox relative to Casp8flox/flox mice might ac-

Figure 3. tr-AMs in LysMCrecasp8flox/flox and cd11cCrecasp8flox/flox mice have preserved expression of casp8, suggesting they escape cre-mediated recombination. (A) CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice have a normal number of TR-AMs in a steady state (n = 5 mice per group; data are shown as mean ± SEM; one-way ANO VA with Dunnett’s test for multiple comparisons; *, P < 0.05; the experiment was performed three times). (B) Casp8 expression in flow-sorted TR-AMs measured by quantitative PCR (n = 3–5 mice per group; data are shown as mean ± SEM; one-way ANO VA with Dunnett’s test for multiple comparisons; **, P < 0.01; the experiment was performed two times). (C and D) Bone marrow from CD11cCreCasp8flox/flox mice fails to recon-stitute the alveolar niche 8 wk after irradiation, as indicated by ongoing recruitment of the immature Mo-AMs with low expression of Siglec F. Repopulation of the alveolar niche was rescued in CD11cCreCasp8flox/floxRIPK3−/− mice (n = 4–5 mice per group; data are expressed as mean ± SEM; one-way ANO VA with Dunnett’s test for multiple comparisons; ****, P < 0.0001). (E) Alveolar macrophages derived from bone marrow from CD11cCreCasp8flox/flox mice fail to reconstitute the alveolar niche 8 wk after irradiation, as shown by decreased expression of Siglec F on alveolar macrophages. Mice were lethally irradiated, followed by reconstitution with bone marrow from control mice (Casp8flox/flox) or bone marrow from mice with deletion of Casp8 in cells expressing CD11c (CreCD11cCasp8flox/flox), either alone or in combination with bone marrow from wild-type mice (50% mixture of the two genotypes). Monocytes, interstitial macrophages, and alveolar macrophages were identified by flow cytometry of lung homogenates 8 wk later (CD45.1 or CD45.2 mice were used for lineage tracing). Although ∼50% of monocytes in both Casp8flox/flox and CreCD11cCasp8flox/flox mice were wild-type cells, the majority of interstitial macrophages and alveolar macrophages in mice reconstituted with CreCD11cCasp8flox/flox bone marrow, but not Casp8flox/flox mice, were of wild-type origin, suggesting the loss of Casp8 during differentiation results in a selective disadvantage in the differentiation of monocytes in to alveolar macrophages; data are shown as mean ± SEM. (F) Cells that escaped Cre-mediated recombination behave similarly to wild-type cells during fibrosis (day 14) with <1% differentially expressed genes (DEG). Volcano plot showing number of differentially expressed genes (red, FDR q value <0.05, n = 2–5 mice per group).

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count for the protection these mice exhibited against bleo-mycin-induced fibrosis, we compared the transcriptomes of sorted naive TR-AMs from Casp8flox/flox, CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice during fibrosis (day 14) and found that <1% of transcripts were differentially expressed (Fig.  3  F), suggesting that the protection we observed in CD11cCreCasp8flox/flox and LysMCreCasp8flox/flox mice against fibrosis was attributable to the loss of Mo-AMs. To exclude the possibility that TR-AMs also contribute to the develop-ment of fibrosis, we selectively depleted TR-AMs by admin-istering liposomal clodronate intratracheally 3 d before the administration of bleomycin. The administration of liposomal clodronate effectively depleted TR-AMs without inducing the recruitment of neutrophils (Fig.  4, A and B). Although TR-AMs were reduced in intratracheal liposomal clodro-nate–treated animals over the course of bleomycin-induced fibrosis, there was no difference in the recruitment of Mo-AM, interstitial macrophages or in the severity of fibrosis (Fig. 4, C–G), suggesting TR-AMs do not contribute to the development of fibrosis in this model.

Our data suggest that Mo-AMs contribute dispropor-tionately to the development of lung fibrosis. To better un-derstand the function of these cells during lung fibrosis, we flow-sorted monocytes, interstitial macrophages, Mo-AMs, and TR-AMs 14 and 19 d after the administration of bleo-mycin and analyzed their gene expression using RNA-seq (Fig. 5 A). Principal-component analysis (PCA) of the dif-ferentially expressed genes showed clustering of the repli-cates according to cellular populations, suggesting that flow cytometry identifies differences in cellular phenotypes that can be reproducibly detected at the level of the transcrip-tome (Fig.  5  B; Fig. S3, A and B; and Table S1). We then performed k-means clustering of the entire transcriptional dataset (12,968 genes), which revealed five distinct clusters of genes (Fig. 5 C, Fig. S3 C, and Tables S2 and S3). Clusters I and II contained core gene signatures that were progres-sively up-regulated and down-regulated during monocyte to alveolar macrophage differentiation, respectively. Genes in these clusters were similar to those others have associated

with monocyte to alveolar macrophage differentiation during homeostasis (Fig. S3 G; Lavin et al., 2014; Schneider et al., 2014). Genes in clusters IV and V were up-regulated early and late during fibrosis, respectively. Early genes (cluster IV) included those involved in biosynthetic pathways, whereas later genes (cluster V) included those involved in epigenetic regulation of gene expression, consistent with the described epigenetic regulation of alveolar macrophage differentiation in the lung (Lavin et al., 2014). These data show that mono-cyte to alveolar macrophage differentiation unfolds slowly over the course of fibrosis.

Because Mo-AMs substantially outnumber TR-AMs during the development of fibrosis, it is possible that both populations respond to fibrosis similarly, but Mo-AMs pre-dominate numerically. To address this question, we compared the expression of profibrotic genes in Mo-AMs and TR-AMs during the development of fibrosis. Cluster III included genes and pathways that are not known to be expressed in alveo-lar macrophages during homeostasis, are not associated with processes involved in cellular differentiation, and are associ-ated with fibrosis (Fig. 5 C). These genes were differentially expressed in monocyte-derived cells (interstitial macrophages and Mo-AMs compared with TR-AMs). We performed pair-wise comparisons of Mo-AMs, interstitial macrophages, and TR-AMs collected 14 or 19 d after bleomycin with naive TR-AMs (Fig. 6 A and Fig. S4 A). 14 d after the adminis-tration of bleomycin, we identified 3,232 genes differentially expressed between TR-AMs and naive TR-AMs and 5,039 genes differentially expressed between Mo-AMs and naive TR-AMs (FDR P < 0.05; Fig. 6 A). Functionally similar genes were differentially expressed 19 d after the administration of bleomycin, but the number of differentially expressed genes was smaller (Fig. S4 A). Gene ontology analysis of the differ-entially expressed genes revealed pathways involved in cell adhesion, cell migration, and fibrosis (Fig. 6, B and C; and Fig. S4 B). Indeed, of the 387 genes in cluster III that were differ-entially expressed in Mo-AMs compared with TR-AMs, 130 were linked to fibrosis in PubMed (Fig. 6 D). For 29 of the genes, other groups have reported that mice lacking the gene

Figure 4. depletion of tr-AMs does not protect from bleomycin-induced lung fibrosis. (A and B) Intratra-cheal administration of 50 μl clodronate-loaded liposomes (Clo-lip) results in nearly complete depletion of TR-AMs 3 d later without recruitment of neutrophils. (C and D) Mice with depleted TR-AMs showed the same severity of bleo-mycin-induced lung fibrosis, as indicated by collagen levels (C) and static lung compliance (D) 21 d later. (E–G) Numbers of TR-AMs (E), Mo-AMs (F), and interstitial macrophages (IMs; G) did not differ between the clodronate-loaded li-posome–treated and control (PBS) groups (n = 5–6 mice per group; data are expressed as mean ± SEM; one-way ANO VA with Dunnett’s test for multiple comparisons; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; the experiment was performed one time).

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were protected against fibrosis in mouse models, including lung (Moore et al., 2006; Flechsig et al., 2010; Riteau et al., 2010; Aschner et al., 2014; Shibata et al., 2014; Tan et al., 2014; Vuga et al., 2014; Agassandian et al., 2015; Brodeur et al., 2015; Wang et al., 2015; Lv et al., 2016), heart (Li et al., 2009; Fan et al., 2014; Nishikido et al., 2016; Toba et al., 2016), liver (Seki et al., 2009; Kim et al., 2010; Madala et al., 2010; Fiorotto et al., 2016; Marí et al., 2016), kidney (Grgic et al., 2009; Lai et al., 2014; Yan et al., 2016), bone (Ishizuka et al., 2011), eye (Chan et al., 2013), muscle (Sinadinos et al., 2015), and skin (Wang et al., 2015). Genes exclusively expressed in TR-AMs included those involved in lipid metabolic processes and blood microparti-cle formation (Fig. 6 B). We performed a functional network analysis of the top 100 differentially expressed genes in cluster III using DAV ID and Fgnet tools (Fontanillo et al., 2011; Aibar et al., 2015). This analysis revealed 14 highly overlapping me-tagroups with a core set of hub genes implicated in fibrotic signaling processes (e.g., Adam8, Arg1, Apoe, Itga6, Mfge8, Mmp12, Mmp13, Mmp14, and Pdgfa; Fig. 6 E and Table S4).

Macrophage polarization refers to distinct sets of in-flammatory (M1) or fibrotic (M2) genes that are expressed by macrophages in cell culture that are first induced toward differentiation and then treated with LPS and IFN-γ or IL-4, respectively (Martinez and Gordon, 2014; Murray et al., 2014). It has been suggested that polarization of alveolar mac-rophages toward a profibrotic or “M2” phenotype contributes to the development of fibrosis (Murray et al., 2010; Lar-son-Casey et al., 2016). Our transcriptional data allowed us to directly test this hypothesis by examining the expression of 20 “M1” and 44 M2 genes during bleomycin-induced lung fi-brosis (Misharin et al., 2014). Interestingly, we found that both Mo-AMs and TR-AMs up-regulated M1 and M2 genes in response to bleomycin without a discernible shift in gene ex-pression toward a M1 or M2 phenotype in either cell popula-tion. When compared with naive TR-AMs, 25% of M1 genes and 18% of M2 genes were differentially higher in intersti-tial macrophages and Mo-AMs during fibrosis (Fig. 6 F and Fig. S4 C). In addition, many M2 genes were higher in naive

Figure 5. tr-AMs and Mo-AMs differ in their response to bleomycin-induced lung fibrosis. (A–C) Transcriptional profiling of macrophage populations over the course of bleo-mycin-induced lung fibrosis. (A) Experimental outline. (B) Principal-component analysis (PCA) of the transcriptomes of FACS-sorted monocytes, interstitial macrophages (IM), Mo-AMs, and TR-AMs during the course of bleomycin-induced lung fi-brosis shows that a significant fraction of the variance in the dataset can be explained by principal component 1 (PC1; 43.7%, differentiation) and principal component 2 (PC2; 17.5%, fibro-sis time course). PCA was performed on differentially expressed genes identified from a generalized linear model to perform an ANO VA-like test for differential expression between any con-ditions in the dataset (FDR step-up procedure q-value < 0.05). (C) k-means clustering of all identified genes revealed clusters of genes associated with monocyte into alveolar macrophage dif-ferentiation (clusters I and II), genes differentially expressed over the course of bleomycin-induced lung fibrosis (clusters IV and V), and genes specifically up-regulated in IM and Mo-AMs, but not in monocytes or TR-AMs (cluster III; optimal k of 5 determined from within-group and between-group sum of squares analysis; Fig. S3 C). The characteristic genes and GO processes associated with each cluster are shown on the left and the right sides, corre-spondingly (see Table S3).

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Figure 6. Monocyte-derived and tissue-resident alveolar macrophages differ in their response to bleomycin-induced lung injury. (A) Volcano plots demonstrating the number of differentially expressed genes (DEG) for the selected comparisons 14 d after instillation of bleomycin (FDR step-up procedure q-value < 0.05). (B) Comparison of DEG and associated GO processes reveals shared and unique response between Mo-AMs and TR-AMs. (C) Hierarchical clustering of the 2708 DEG shared by Mo-AMs and TR-AMs during the response to bleomycin-induced lung fibrosis. (D) Association between genes in cluster III and fibrosis. Differentially expressed genes from cluster III were taken; term “AND Fibrosis” was added to the gene names and the result-ing term was used as input term in the PubMed search engine to search abstracts and full text using an in-house Python script. The abstracts were then manually reviewed for evidence of a genetic association with fibrosis (gene knockouts were protected from fibrosis or transgenic overexpression of the gene increased fibrosis). Of the 387 differentially expressed genes in cluster III (FDR q-value < 0.05), 203 were linked with fibrosis in PubMed and 23 were causally related to the development of fibrosis in different organs (as indicated by images in figure) in genetic mouse models. The numbers in parenthesis are PubMed IDs. (E) Functional gene network analysis was performed on the top 100 of differentially expressed genes between Mo-AMs and TR-AMs from cluster III using GeneTerm Linker and visualized using FGNet tool. White circles indicate hub genes that belong to the multiple metagroups. For metagroup annotations, see Table S4. (F) Heat map of M1/M2 genes that were differentially expressed in our dataset (FDR step-up q-value < 0.05; see also Fig. S4 C).

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TR-AMs than in TR-AMs isolated during fibrosis (Pparg, Tgm2, Cebpb, and Tgfb1). Collectively, these results show that Mo-AMs and TR-AMs alter their expression of both M1 and M2 genes during bleomycin-induced fibrosis and argue against an exclusive role for macrophage polarization toward a M2 phenotype to explain their contribution.

It is not known whether the alveolar macrophage pool is restored after the resolution of injury through the persistence of Mo-AMs recruited to the lung or whether TR-AMs pro-liferate to repopulate the alveolar macrophage pool. Bleomy-cin-induced fibrosis in young mice slowly resolves over 60 d (Hecker et al., 2014). Accordingly, we treated shielded bone marrow chimeric mice with bleomycin and measured the ratio between Mo-AMs and TR-AMs 10 mo later. Extend-ing the findings from several groups (Guilliams et al., 2013; Hashimoto et al., 2013; van de Laar et al., 2016), we found that TR-AMs were remarkably stable, constituting >95% of alveolar macrophages at 14 mo of age (Fig. 7, A and B). In contrast, in mice that received bleomycin at 8 wk post–bone marrow transfer (4 mo of age), 10 mo later, ∼50% of the alveolar macrophages were monocyte derived. To determine whether this finding was unique to bleomycin, we treated a separate cohort of shielded bone marrow chimeric mice with influenza A (A/WSN/33) virus, at a dose that was associated with severe injury but was not lethal, and obtained similar results (Fig.  7 B). This finding suggests that a severe injury early in life can permanently reshape the alveolar macrophage landscape with respect to its developmental origins.

Next, we sought to determine whether the differences in gene expression between TR-AMs and Mo-AMs during fibrosis persisted over the life span. 10 mo after bleomycin treatment or influenza A infection, TR-AMs and Mo-AMs expressed similar levels of Siglec F and were no longer dis-tinguishable by flow cytometry (Fig. 7 C). A comparison of the transcriptomes of TR-AMs and Mo-AMs 10 mo after bleomycin administration revealed only 330 differentially ex-pressed genes (Fig. 7 D and Table S5).

The role of immune cell infiltration in the development of human lung fibrosis remains controversial. Therefore, we used flow cytometry to assess the number of alveolar macro-phages in distal lung tissue obtained from the explanted lung, and a biopsy of the donor lung, in patients with lung fibrosis undergoing lung transplantation. We observed an expansion of alveolar macrophages in lung tissue samples from patients with fibrosis compared with the donor controls (Fig.  8, A and B; Bharat et al., 2016; Desch et al., 2016; Yu et al., 2016). Expansion of the alveolar macrophage pool was confirmed by immunostaining lung sections with the alveolar macro-phage markers CD206 and CD169 (Fig. 8 C). As we found that Mo-AMs are causally linked to the development of lung fibrosis in response to bleomycin and Ad-TGF-β, we sought to determine whether the human homologues of the mouse profibrotic genes we identified in Mo-AMs during bleomy-cin-induced fibrosis (Fig. 5 C, cluster III) were up-regulated in alveolar macrophages isolated from the lungs of patients with lung fibrosis. We flow-sorted alveolar macrophages from

Figure 7. Monocyte-derived alveolar mac-rophages persist after the resolution of lung injury and fibrosis. (A) Schematic of the experi-mental approach. Shielded bone marrow chimeras were harvested 10 mo after challenge with either bleomycin or with influenza A virus. (B) Percentage of cells identified as TR-AMs (CD45.2) and Mo-AMs (CD45.1) in untreated shielded chimeric mice, mice that received bleomycin, or mice infected with 100 PFU influenza A virus (A/WSN/33) as young adults (n = 3–5 mice per group; data are expressed as mean ± SEM). (C and D) 10 mo after the initial challenge, Mo-AMs and TR-AMs are no longer dis-tinguishable by flow cytometry, and transcriptional profiling revealed only 330 differentially expressed genes (DEG; FDR step-up procedure q-value < 0.05; see Table S5). (E) Schematic illustration of the dif-ferential role of TR-AMs and Mo-AMs during the different stages of the lung injury and fibrosis.

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patients with end-stage lung fibrosis attributed to one of sev-eral etiologies (idiopathic pulmonary fibrosis, scleroderma- associated interstitial lung disease, polymyositis-associated interstitial lung disease, and mixed connective tissue disease) and alveolar macrophages from a small biopsy specimen of normal lung obtained from the donor, and we analyzed their transcriptome using RNA-seq. Of the genes differentially ex-pressed in cluster III (Table S2), homologues of 61 genes were also differentially expressed between alveolar macrophages isolated from patients with lung fibrosis compared with do-nors: 51 were up-regulated in patients with lung fibrosis, in-cluding APOE, ITGA6, FGFR1, MMP12, MMP14, PDPN, and SPA RC (Fig. 8 D; normalized counts in Table S6), which suggests the existence of common profibrotic pathways in human and mouse macrophages as well as highly conserved profibrotic alveolar macrophage signature across heteroge-neous clinical entities. Several genes that were up-regulated in mouse Mo-AMs were down-regulated in patients with lung fibrosis, and some of these genes have been implicated in lung repair after injury (e.g., CD163, MFGE8, LYVE1, and C1QB). A pathogenic role for alveolar macrophages and other inflammatory cells has been dismissed because a human clinical trial showed the administration of corticosteroids was shown to have no effect or slightly worsened clinical outcomes in patients with idiopathic pulmonary fibrosis (Raghu et al., 2012). To determine whether corticosteroids targeted alveolar macrophages during the development of fibrosis, mice were treated with dexamethasone (equivalent prednisone dose of 1 mg/kg/day, intraperitoneal) beginning simultaneous with the administration of bleomycin. The severity of bleo-mycin-induced lung fibrosis in dexamethasone- and sham- treated mice was similar (Fig. S5, A–C). Dexamethasone treat-ment did not significantly alter the depletion of TR-AMs or the recruitment of Mo-AM during fibrosis (Fig. S5, D and E), consistent with the suggestion that corticosteroid treatment has little or no effect on the population of macrophages.

dIscussIonWe used a genetic lineage tracing system to show that Mo-AMs and TR-AMs play distinct roles during the devel-opment of lung fibrosis. In common mouse models of lung fibrosis, the deletion of Mo-AM after their recruitment to the lung markedly attenuated the severity of fibrosis, whereas the deletion of TR-AM had no effect on fibrosis severity. These findings are bolstered by transcriptomic profiles of monocyte and macrophage populations over the course of bleomycin-induced fibrosis, which reveal substantial differ-ences in gene expression between Mo-AMs and TR-AMs over the course of lung fibrosis. Our findings suggest revi-sion of our consideration of alveolar macrophages as a sin-gle population of cells in the design of macrophage-targeted therapies in lung fibrosis.

Our transcriptomic data of flow-sorted monocyte and macrophage populations from lung homogenates suggest that monocyte to alveolar macrophage differentiation represents

a continuous down-regulation of genes typically expressed in monocytes and up-regulation of genes expressed in alveo-lar macrophages. These findings suggest that monocytes give rise to interstitial macrophages, which in turn give rise to Mo-AMs, and are consistent with published data suggesting that monocyte to macrophage differentiation results from epigenetic reprogramming driven by factors present in the local microenvironment (Landsman and Jung, 2007; Lavin et al., 2014). We observed that the protection against bleo-mycin-induced lung fibrosis in both CreCD11cCasp8floxflox and CreLysMCasp8floxflox mice relative to Casp8floxflox controls. This protection was associated with preserved numbers of inter-stitial macrophages but a marked reduction in Mo-AMs. Caspase-8 initiates apoptosis through caspase-3/7 activation and blocks necroptosis via suppression of RIPK1–RIPK3 signaling (Hutcheson and Perlman, 2008; Oberst et al., 2011). The genetic loss of RIPK3 restored the population of Mo-AMs, and sensitivity to bleomycin induced fibrosis in caspase-8–deficient mice, suggesting these cells undergo necroptosis as they differentiate from interstitial macrophages to Mo-AMs. These results are consistent with previous studies that have demonstrated the importance of monocytes in the development of bleomycin-induced fibrosis (Gibbons et al., 2011; Larson-Casey et al., 2016) but represent an important advance with therapeutic implications. Systemic depletion of monocytes or inhibition of pathways required for monocyte migration to sites of tissue injury is necessarily limited by toxicity, as monocyte migration is required to maintain some tissue resident macrophage populations (e.g., in the gut) and for the systemic maintenance of dendritic cells (Lavin et al., 2015). Our data suggest that targeting pathways required for alveolar macrophage differentiation after monocytes have begun the process of differentiation in the lung can prevent fibrosis. Because many of these pathways are exclusively im-portant for alveolar macrophages (Nakamura et al., 2013; Lavin et al., 2014; Schneider et al., 2014; Sennello et al., 2017), targeting them is predicted to slow the development of Mo-AMs without affecting monocyte or macrophage popu-lations in other tissues.

Although TR-AMs increased the expression of some profibrotic genes during fibrosis, this response was substan-tially muted relative to Mo-AMs. We found that depletion of TR-AMs using intratracheal liposomal clodronate before the administration of bleomycin had no effect on fibrosis, sug-gesting they are dispensable for the development of fibro-sis in this model. Furthermore, the significant protection we observed in CreCD11cCasp8floxflox and CreLysMCasp8floxflox mice cannot be attributed to deletion of caspase-8 in TR-AMs, as we found the TR-AMs in these mice had preserved ex-pression of Casp8, suggesting they escaped genetic recombi-nation. Indeed, there were few detectable differences in the transcriptomes of TR-AMs from CreCD11cCasp8floxflox and CreLysMCasp8floxflox mice compared with Casp8floxflox controls. Competitive chimera experiments and bone marrow recon-stitution with CreCD11cCasp8floxfloxRIPK3−/− animals suggest

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that in the absence of caspase-8, monocyte-derived cells (likely interstitial macrophages) undergo necroptosis as they differentiate into Mo-AMs. Although this suggests that RIPK is activated during alveolar macrophage differentiation, we were unable to identify a role for this pathway in fibrosis, as the transcriptomes of CreCD11cCasp8floxfloxRIPK3−/− Mo-AMs

were similar to controls. Interestingly, although interstitial macrophages demonstrated even higher levels of profibrotic gene expression than Mo-AMs and their numbers were sim-ilar in CreCD11cCasp8floxflox and CreLysMCasp8floxflox mice com-pared with Casp8floxflox controls, protection against fibrosis was observed in the Casp8-deficient mice. This might reflect the

Figure 8. Alveolar macrophages are expanded in patients with lung fibrosis and exhibit a profibrotic gene expression signature similar to mouse Mo-AMs during lung fibrosis. (A–C) The population of alveolar macrophages is increased in patients with fibrotic lung disease. At the time of lung transplantation, small distal lung biopsy specimens were harvested from the donor lung (n = 16) and the recipient lung (n = 10) and processed for flow cytometry and immunohistochemistry. (A) Alveolar macrophages were identified as CD206+CD169+HLA-DR+ cells out of all hematopoietic CD45+ cells from donor lung tissue and from patients with lung fibrosis (Mann–Whitney test; data are expressed as mean ± SD). (B) The same analysis was repeated after excluding neutrophils (Mann–Whitney test; data are expressed as mean ± SD). (C, top) Masson’s trichrome staining; bar, 500 µm. (C, middle) Immunohisto-chemistry for CD169; bar, 50 µm. (C, bottom) Immunohistochemistry for CD206; bar, 50 µm. Representative images from healthy donor, patients with idio-pathic pulmonary fibrosis (IPF), systemic sclerosis-associated interstitial lung disease (SSc-ILD), mixed connective tissue disease (MCTD), and hypersensitivity pneumonitis (HP) are shown. (D) Of the differentially expressed genes in cluster III (Table S2), homologues of 61 were differentially expressed (FDR step-up procedure q-value < 0.05) between alveolar macrophages isolated from human fibrotic lung explants compared with donor lungs; 51 were up-regulated in patients with lung fibrosis, and 10 were down-regulated.

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relatively low abundance of interstitial macrophages in com-parison to Mo-AMs, a presumed difference in their life span, or their anatomical localization in the interstitium versus al-veolar space, respectively.

It has long been known that complete depletion of al-veolar macrophages with ionizing radiation results in their replacement by Mo-AMs that closely resemble TR-AMs (Hashimoto et al., 2013; Lavin et al., 2014; van de Laar et al., 2016). Although others have previously shown that monocytes recruited during the tissue injury can give rise to long-living self-renewing tissue-resident-like cells in the skin (Merad et al., 2002), it is not known whether Mo-AMs recruited to the lung during injury persist in the lung or whether the alveolar macrophage population is restored via proliferation of TR-AMs. We found that in the absence of injury, TR-AMs were a remarkably stable population, with <5% of cells originating from the bone marrow in 14-mo-old mice. In contrast, 1 yr after the administration of bleomy-cin, when fibrosis has completely resolved, ∼50% of alveolar macrophages were monocyte derived. Although Mo-AMs and TR-AMs showed marked differences in the expression of profibrotic genes during the development of fibrosis, 1 yr after fibrosis, they were indistinguishable by flow cytometry and showed only small differences in their transcriptome. Our design does not allow us to determine whether these retained Mo-AMs harbor an epigenetic memory of the inflammatory and fibrotic environment in which they were generated that is distinct from TR-AMs and respond differently to subse-quent environmental challenges (Ostuni et al., 2013). Never-theless, these results show that environmental exposures early in life can induce heterogeneity in the ontologic origins of alveolar macrophages, offering a possible mechanism to ex-plain the enhanced and persistent fibrosis reported with re-peated doses of bleomycin and during aging (Degryse et al., 2010; Hecker et al., 2014).

In lung transplant recipients examined up to 5 yr after transplantation, investigators reported that the majority of alveolar macrophages originated from the donor, suggesting the presence of a stable population of TR-AMs in humans analogous to those found in mice (Nayak et al., 2016). The decline in lung function in patients with fibrosis is not con-tinuous, and step declines are often preceded by the devel-opment of “exacerbations,” or an acute or subacute clinical worsening of shortness of breath and hypoxemia accompa-nied by inflammatory infiltrates on computed tomography imaging of the chest that suggest a role for immune cells in the pathogenesis of pulmonary fibrosis (Collard et al., 2007). However, a multicenter clinical trial of corticosteroids for the treatment of pulmonary fibrosis failed to show benefit, with a nonsignificant trend toward harm (Raghu et al., 2012). As a result of this trial and studies reporting that lung inflam-mation is sometimes worse in mice with genetic mutations that prevent the development of fibrosis, many concluded that inflammation is dispensable for the development of fibrosis (Munger et al., 1999; Budinger et al., 2006; Rock et al., 2011).

Our data challenge these findings. We detected a substantial expansion in macrophages using newly described flow cy-tometry markers in distal lung tissue from a small number of patients with pulmonary fibrosis compared with normal lungs from the donor. Transcriptional profiling of these cells revealed substantial overlap in the expression of human ho-mologues of the profibrotic genes up-regulated in Mo-AMs in mice exposed to bleomycin. This led us to examine the effect of corticosteroids on macrophages in lung fibrosis. Sim-ilar to the human trial, corticosteroids had no effect on the se-verity of bleomycin-induced lung fibrosis; however, we found that neither the TR-AM nor the Mo-AM population was affected by steroid treatment.

Our study has several limitations. Although the ap-proach we used is a powerful tool to identify the sequence of transcriptional modifications that develop during monocyte to macrophage differentiation during disease, pulsed lineage tracing techniques will be required to follow an individual population of monocytes during their differentiation into Mo-AMs (Paul et al., 2015). Second, our flow cytometry ap-proach cannot provide anatomical detail, except when anti-bodies for immunofluorescence or immunohistochemistry are available. For example, it is possible that Mo-AMs that persist in the lung after injury are differentially localized to areas where the microenvironment is subtly abnormal. Third, although our data suggest that the concept of M2 polariza-tion overly simplifies the complex changes in gene expression that occur in both Mo-AMs and TR-AMs over the course of lung fibrosis (Nahrendorf and Swirski, 2016), it is possible that a subpopulation of Mo-AMs with one of these signatures or at different developmental stages contributed dispropor-tionately to the fibrotic gene expression signature. Single-cell RNA-seq analysis will be required to exclude this possibil-ity. Fourthly, the CreLysM and CreCD11c promoters both target dendritic cells. Therefore, although we did not see changes in the numbers of dendritic cells in the lungs of the caspase-8–deficient mice generated using these promoters, we cannot exclude the possibility that some of the effects we observed are attributable to dendritic cells. Finally, although we are un-aware of previous studies showing an expansion of the alveo-lar macrophage pool in patients with lung fibrosis or previous studies of transcriptional profiling of these cells during fi-brosis, we cannot determine whether they play a causal role in disease pathogenesis.

In summary, our results suggest that an appreciation of macrophage heterogeneity has important implications for disease pathogenesis and therapeutic development. Mo-AMs undergo dramatic transcriptional changes as they differentiate in the injured lung through a process that unfolds slowly over time. Mo-AMs express consistently higher levels of proin-flammatory and profibrotic genes than TR-AMs, and selective depletion of Mo-AMs, but not TR-AMs, ameliorates the se-verity of lung fibrosis. Profibrotic gene expression in Mo-AMs harvested from mice during fibrosis is homologous to genes differentially regulated in alveolar macrophages from patients

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with lung fibrosis. These results challenge the consideration al-veolar macrophages during lung fibrosis as a single population of M2 polarized macrophages and suggest that selectively tar-geting Mo-AMs after their commitment to an alveolar mac-rophage fate may ameliorate fibrosis without the off-target effects of therapies that deplete circulating monocytes. Our finding that Mo-AMs persist in the lung after the resolution of injury shows that environmental exposures can permanently change the origins of alveolar macrophages. Determining whether retained Mo-AMs and TR-AMs respond differently to a subsequent injury or differentially change their gene ex-pression signature with age may offer a mechanism to under-stand the increased risk of lung fibrosis with advancing age.

MAterIALs And MetHodsHuman subjectsAll human studies were approved by Northwestern IRB and Department of Defense Human Research Protection Office. All human subjects provided written informed con-sent before enrolment into the study. A biopsy-size piece of the donor lung tissue and a lobe of the explanted recip-ient lung were obtained at the time of the lung transplant. Tissues were processed for flow cytometry and histology as previously described (Bharat et al., 2016). 10 patients with fibrotic lung disease (2 patients with idiopathic pul-monary fibrosis, 5 patients with systemic sclerosis-associated interstitial lung disease, 1 patient with mixed connec-tive tissue disease, 1 patient with interstitial pneumoni-tis, and 1 patient with pneumoconiosis) were included in flow cytometric analysis.

MiceAll mouse procedures were approved by the Institutional Animal Care and Use Committee at Northwestern Univer-sity. The mouse strains C57BL/6, B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) were used. All strains including wild-type mice are bred and housed at a barrier- and specific pathogen–free fa-cility at the Center for Comparative Medicine at Northwest-ern University (Chicago, IL). Colonies are refreshed at least yearly with mice purchased from The Jackson Laboratory. 8–10-wk-old mice were used for all experiments, although some were aged up to 14 mo in our facility. Casp8flox/flox, CD-11cCreCasp8flox/flox, LysMCreCasp8flox/flox, CD11cCreCasp8flox/

floxRIPK3−/−, and LysMCreCasp8flox/floxRIPK3−/− mice were described previously (Cuda et al., 2014, 2015); these mice were backcrossed to C57BL/6 background for >10 genera-tions. Littermate controls were used in all experiments, ex-cept for generation of competitive bone marrow chimeras. All procedures were approved by the Institutional Animal Care and Use committee at Northwestern University. Bleomy-cin-induced lung fibrosis, TGF-β–induced lung fibrosis, and influenza A virus infection (A/WSN/33) were performed and assessed as previously described (Budinger et al., 2006; Misharin et al., 2013; Morales-Nebreda et al., 2014, 2015; Sennello et al., 2017).

tissue preparation and flow cytometryTissue preparation for flow cytometry analysis and cell sorting was performed as previously described (Misharin et al., 2013, 2014; Bharat et al., 2016). Blood was collected into EDTA-con-taining tubes via facial vein bleed (from live animals) or cardiac puncture (from euthanized animals). Whole blood was stained with fluorochrome-conjugated antibodies, and erythrocytes were then lysed using BD FACS lysing solution. For single-cell suspension obtained from tissues erythrocytes were lysed using BD Pharm Lyse, and cells were counted using Countess auto-mated cell counter (Invitrogen); dead cells were discriminated using trypan blue. Cells were stained with eFluor 506 (eBiosci-ence) viability dyes, incubated with FcBlock (BD), and stained with fluorochrome-conjugated antibodies (antibodies, clones, fluorochromes, and manufacturers were described in detail in our previous publications; Misharin et al., 2013, 2014; Bharat et al., 2016). Data were acquired on BD LSR II flow cytometer (for information regarding instrument configuration and anti-body panels, see Misharin et al., 2013, 2014; Bharat et al., 2016). Compensation, analysis and visualization of the flow cytometry data were performed using FlowJo software (Tree Star). “Fluo-rescence minus one” controls were used when necessary to set up gates. Cell sorting was performed at Northwestern Univer-sity RLH CCC Flow Cytometry core facility on SORP FAC SAria III instrument (BD) with the same optical configuration as LSR II, using a 100-µm nozzle and 40 psi pressure.

Bone marrow chimeras and shielded bone marrow chimerasBone marrow chimeras were established by transferring 5 × 106 bone marrow cells isolated from C57BL/6 mice (this strain expresses CD45.2 alloantigen) into 8-wk-old le-thally irradiated (single dose of 1,000 cGy γ-radiation using a Cs-137–based Gammacell-40 irradiator; Nordion) recipient mice (expressing CD45.1 alloantigen). Mice were maintained on autoclaved water supplemented with antibiotics (trimetho-prim/sulfamethoxazole; Hi-Tech Pharmacal) for 4 wk after bone marrow transfer and then switched to normal housing regimen. CD45.2 to CD45.1 bone marrow chimeras were used for experiments 8 wk after bone marrow transfer. 8 wk after bone marrow transfer, >95% of all leukocytes and 100% of monocytes and neutrophils in peripheral blood were of donor origin. Bone marrow chimeras with thoracic shielding were used to assess the origin of pulmonary macrophages (TR-AMs vs. Mo-AMs) and were generated in a manner similar to that previously described (Janssen et al., 2011), with additional modifications (Misharin et al., 2014). To protect TR-AMs from radiation, we applied a uniform lead shield that covered the lungs during irradiation as previously described using a cus-tom-made “Mousotron-5” apparatus. However, we found this approach was limited, as the residual recipient bone marrow in the shielded region results in incomplete chimerism; ∼20% of peripheral blood monocytes are still of recipient origin and cannot be distinguished from tissue-resident macrophages upon recruitment to the lung. This problem was corrected by the administration the myeloablative agent busulfan (30 mg/kg

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body weight; Sigma-Aldrich; Chevaleyre et al., 2013) 6 h after the irradiation, followed 12 h later by bone marrow infusion. 2 mo after the procedure, 100% of all alveolar macrophages in the shielded bone marrow chimeras were of recipient origin (CD45.2), whereas 100% of all circulating monocytes were of donor origin (CD45.1). Bone marrow chimeras with thoracic shielding were maintained on antibiotics for 4 wk as described above and then switched back to the normal housing regimen.

Gene expression profiling (rnA-seq) and bioinformatic analysisIn the overall design, monocyte and macrophage subpop-ulations were isolated from naive, bleomycin-, or influenza A virus–challenged mice at the indicated time points via FACS-sorting on BD FAC SAria III instrument and RNA was extracted for subsequent transcriptomic analysis. For each genotype/condition, two to five biological replicates were used. Libraries for RNA-seq were prepared in 96-well plate format. For the bleomycin time-course experiment, 155 samples were equally distributed between two batches of the libraries preparation (78 and 77 samples), so each group/con-dition would be represented on each plate. For experiments using shielded chimeras with busulfan (40 samples), libraries were prepared as a single batch.

Monocyte and macrophage subpopulations were iso-lated on BD FAC SAria III instrument, 0.5–1.5 × 105 cells were sorted into capture media (PBS, 2% BSA and 0.05% EDTA), immediately spun down and lysed in 350 μl of RLT-plus buffer (QIA GEN) supplemented with β2-mercaptoeth-anol. Total RNA was extracted using RNeasy Plus Mini kit (QIA GEN) and eluted in 35 μl of nuclease free water. RNA quality and quantity were assessed using Bioanalyzer 2100 or TapeStation 4200 instruments (Agilent Technologies). RNA-seq libraries were prepared in 96-well plate format using NEBNext Poly(A) mRNA Magnetic Isolation Mod-ule, NEBNext Ultra RNA Library Prep kit for Illumina and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) according to the standard protocol. 50 ng total RNA was enriched for poly(A) mRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module, the mRNA was subjected to chemical fragmentation in the presence of divalent cations at 94°C for 15 min, and cDNA was gener-ated using random primers and ProtoScript II reverse tran-scription in the presence of mouse RNase inhibitor using the following program: 10 min at 25°C, 15 min at 42°C, and 15 min and 70°C. Second-strand DNA was generated using NEBNext Second Strand Synthesis Enzyme module (DNA polymerase I, RNase H, Escherichia coli DNA ligase) for 60 min at 16°C. The resulting double-stranded DNA was puri-fied using 1.8× volume of SPRI beads (AMPure XP Beads; Beckman Coulter) or HighPrep PCR Beads (MagBio Ge-nomics), eluted in 0.1× TE (Tris/EDTA) buffer, DNA ends were repaired using NEBNext End Prep Enzyme Mix (T4 PNK and T4 DNA polymerase) for 30 min at 20°C and 30 min and 65°C, followed by NEBNext adaptor for Illumina

ligation using Blunt/TA Ligase master mix (T4 DNA ligase) for 15 min at 20°C and incubation with USER enzyme for 15 min at 37°C. After another round of purification using 1× volume of SPRI Beads, adaptor-ligated DNA was amplified using NEBNext Q5 Hot Start HiFi DNA polymerase in the presence of NEBNext multiplex oligos (dual index primers) for 14 cycles. PCR-enriched libraries were purified using a 0.9× volume of SPRI Beads, and their quality was assessed using Bioanalyzer 2100 or TapeStation 4200 instruments. Equimolar amount of libraries were pooled and sequenced on Illumina NextSeq 500 instrument (single end reads) using V1 chemistry high-output 150 cycles sequencing kit (bleomycin time-course experiment) or V2 chemistry 75 cycles high-out-put sequencing kit (aged-shielded bone marrow chimeras).

Computation intensive analysis was performed using “Genomics Nodes” on Northwestern’s High Performance Computing Cluster, Quest (Northwestern IT and Research Computing). Reads were demultiplexed using bcl2fastc, quality was assessed using FastQC, reads were trimmed and aligned to mm10 reference genome using TopHat2, read counts were associated with genes using the GenomicRanges (Lawrence et al., 2013), and differential gene expression was assessed using edgeR (Robinson et al., 2010; McCarthy et al., 2012) R/Bioconductor packages. Genes with less than one normalized read count across at least half of the samples were filtered from all analyses. Pearson correlation matrix and clustering heat maps were built using GENE-E (J. Gould, 2013; GENE.E: Interact with GENE-E from R. R pack-age version 1.12.2; http ://www .broadinstitute .org /cancer /software /GENE -E). Gene ontologies were evaluated using GOrilla (Eden et al., 2007, 2009). The dataset is available on the GEO database under accession number GSE82158.

statistical analysisFor all fibrosis measurements, the data were analyzed using two-way ANO VA for repeated measurements with Bonfer-roni post-test to compare differences between the groups. All analyses were performed using GraphPad Prism version 7.00 (GraphPad Software). Data are shown as means ± SEM.

online supplemental materialFig. S1 shows the gating strategy used to isolate subsets of monocytes and macrophages during the course of bleomy-cin-induced lung fibrosis. Fig. S2 shows data demonstrating protection from Ad-TGFb-induced lung fibrosis in Casp8-de-ficient mice. Fig. S3 shows global transcriptome differences be-tween populations of macrophages and relevant GO processes. Fig. S4 (related to Fig. 6) shows differences between TR-AMs and Mo-AMs at day 19 of bleomycin-induced lung fibrosis and absence of evidence supporting clear M1/M2 pattern of macrophage polarization in vivo. Fig. S5 shows glucocorticoids have no effect on the course of bleomycin-induced lung fibro-sis. Table S1 (related to Fig. 5 and Fig. S3) contains Pearson’s correlation coefficients for transcriptomes of the individual macrophage populations. Table S2 (related to Fig. 5) contains

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k-means cluster assignment. Table S3 (related to Fig. 5 and Fig. S3) contains GO processes and statistics for each cluster. Table S4 (related to Fig. 6) contains results of Fgnet enrichment. Table S5 (related to Fig. 7) contains list of differentially expressed genes between TR-AMs and Mo-AMs 10 mo after bleomy-cin-induced lung fibrosis. Table S6 (related to Fig. 8) contains normalized gene counts for human homologues of the mouse genes from cluster III. Tables S1–S6 are included as Excel files.

AcknoWLedGMentsA.V. Misharin is supported by National Institutes of Health (NIH) National Institute of Arthritis and Musculoskeletal and Skin Diseases grant AR061593, an American Tho-racic Society/Scleroderma Foundation research grant, Department of Defense grant PR141319, and a BD Bioscience immunology research grant. P.A. Reyfman is sup-ported by Northwestern University's Lung Sciences Training Program (5T32 HL076139-13). C.M. Cuda is supported by NIH grant AR064313. S. Chiu is supported by Northwestern University’s Transplant Surgery Scientist Training Program (NIH grant T32 DK077662) and the American Society for Transplant Surgery Foundation. A. Bharat is supported by NIH grant HL125940 and matching funds from Thoracic Sur-gery Foundation, a research grant from the Society of University Surgeons, and an American Association of Thoracic Surgery John H. Gibbon Jr. Research Scholarship. B.D. Singer is supported by NIH grant HL128867 and the Parker B. Francis Research Opportunity Award. J.I. Sznajder is supported by NIH grants AG049665, HL048129, HL071643, and HL085534. G. Multlu is supported by NIH grants ES015024 and ES025644. K. Ridge is supported by NIH grants HL079190 and HL124664. G.R.S. Budinger is supported by NIH grants ES013995, HL071643, and AG049665; Veterans Administration grant BX000201, and Department of Defense grant PR141319. H. Per-lman is supported by NIH grants AR064546, AG049665, and HL134375 and funds provided by Mabel Greene Myers Chair. The Northwestern University Flow Cytometry Facility and Center for Advanced Microscopy are supported by a National Cancer In-stitute cancer center support grant P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. The Genomics Computing Cluster is jointly supported by the Feinberg School of Medicine, the Center for Genetic Medicine, and Feinberg’s Department of Biochemistry and Molecular Genetics, the Office of the Provost, the Office for Research, and Northwestern Information Technology and maintained and developed by Feinberg IT and Research Computing Group.

The authors declare no competing financial interests.Author contributions: A.V. Misharin contributed to conceptualization, method-

ology, validation, formal analysis, investigation, resources, data curation, writing, visu-alization, supervision, project administration, and funding acquisition. L. Morales-Nebreda contributed to conceptualization, methodology, validation, formal analysis, investigation, data curation, writing, and visualization. P.A. Reyfman contrib-uted to conceptualization, methodology, software, validation, formal analysis, investi-gation, data curation, writing, visualization, and supervision. T.J. Yacoub contributed to conceptualization, methodology, software, validation, formal analysis, data curation, writing, and visualization. C.M. Cuda contributed to conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, and writing. H. Ab-dala-Valencia contributed to investigation, resources, data analysis, data curation, writing, visualization, and project administration. J.M. Walter contributed to formal analysis, investigation, data curation, writing, and visualization. A.C. McQuattie-Pimen-tel contributed to methodology, validation, formal analysis, investigation, data cura-tion, and writing. C. Chen contributed to methodology, validation, formal analysis, investigation, and data curation. N. Joshi contributed to methodology, validation, formal analysis, investigation, and data curation. K.J.N. Williams contributed to meth-odology, validation, formal analysis, investigation, and data curation. M. Chi contrib-uted to methodology, validation, formal analysis, investigation, and data curation. S. Chiu contributed to methodology, validation, formal analysis, investigation, data cura-tion, and writing. F.J. Gonzalez-Gonzalez contributed to methodology, validation, for-mal analysis, investigation, and data curation. K. Gates contributed to methodology, validation, formal analysis, investigation, and data curation. A.P. Lam contributed to methodology, validation, formal analysis, investigation, and data curation. T.T. Nichol-son contributed to investigation and data curation. P.J. Homan contributed to meth-odology, validation, formal analysis, investigation, and data curation. S. Soberanes contributed to methodology, validation, formal analysis, investigation, and data cura-tion. S. Dominguez contributed to methodology, validation, and investigation. V.K.

Morgan contributed to methodology, validation, investigation, and data curation. R. Saber contributed to validation, formal analysis, investigation, and data curation. A. Shaffer contributed to validation, formal analysis, investigation, and data curation. K.R. Anekalla contributed to methodology, software, validation, formal analysis, investiga-tion, data curation, writing, and visualization. S.A. Marshall contributed to methodol-ogy, formal analysis, and investigation. M. Hinchcliff contributed to resources, data curation, writing, and supervision. A. Bharat contributed to resources, data curation, writing, and supervision. S.M. Bhorade contributed to resources, data curation, writing, and supervision. B.D. Singer and S. Berdnikovs contributed to methodology, validation, formal analysis, writing, and visualization. E.T. Bartom contributed to methodology, validation, software, and formal analysis. W.E. Balch contributed to conceptualization, resources, data curation, writing, supervision, and project administration. R.I. Morimoto contributed to conceptualization, resources, data curation, writing, and supervision, project administration. J.I. Sznajder contributed to conceptualization, resources, data curation, writing, supervision, and project administration. N.S. Chandel contributed to conceptualization, data curation, writing, and supervision. G.M. Mutlu contributed to conceptualization, resources, data curation, writing, and supervision. M. Jain contrib-uted to conceptualization, data curation, writing, and supervision. C.J. Gottardi con-tributed to conceptualization, resources, writing, supervision, and project administration. K.M. Ridge contributed to conceptualization, resources, writing, super-vision, and project administration. N. Bagheri contributed to conceptualization, meth-odology, formal analysis, resources, data curation, writing, and visualization. A. Shilatifard contributed to methodology, resources. GR.S. Budinger contributed to con-ceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing, visualization, supervision, project administration, and funding acqui-sition. H. Perlman contributed to conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing, visualization, supervision, project administration, funding acquisition.

Submitted: 19 December 2016

Revised: 2 April 2017

Accepted: 25 May 2017

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